Cartridge, cartridge reader and method for preventing reuse of the cartridge

ABSTRACT

A disposable fluidic cartridge has a recess therein. A mechanically-actuated mechanism is disposed within the recess for effecting an operation of the fluidic cartridge. The cartridge further comprises a machine-readable identification code on a breakable label covering at least a portion of the recess such that the portion of the breakable label covering the recess may be broken upon insertion of a mechanical actuator into the recess to actuate the mechanically-actuated mechanism. This renders the identification code unreadable and prevents reuse of the cartridge.

FIELD

The present invention relates to a disposable fluidic cartridge, acartridge reader and an associated method for preventing reuse of thedisposable cartridge. In particular, the present invention relates to acartridge, reader and method for performing a diagnostic test on thecartridge wherein the test is only commenced is an identification codeis detected.

BACKGROUND

Sample preparation and analysis presents many logistical problems.Conventionally, many medical samples (such as blood, saliva, urine andswab eluate) are provided to a doctor, for example a generalpractitioner doctor (GP) or a principle care physician (PCP), in a localsurgery without the equipment necessary to analyse the sample. Hence,the sample must be sent to a laboratory where the sample is analysed.The test results must then be collated and returned to the GP to analysethe results and make a diagnosis. This approach is inadequate. Firstly,there is a significant risk that a sample is lost in transit ormismatched with the wrong patient. Moreover, whilst recent developmentsin technology have reduced the overall time taken to conduct the test,the delay involved in sending the sample to a laboratory isunsatisfactory.

Nevertheless, analytical systems of the kind found in laboratories arecomplex and it is often difficult to provide sufficient amounts of puretargets from source samples to reliably perform downstream analyticalassays. This typically prohibits local GP surgeries from being able tocarry out such tests on site.

However, in recent years efforts have been made to reduce the scale ofthe analytical systems to make tests faster and simpler to run, andrequire smaller quantities of sample. For instance, “laboratory on achip” (LOC) devices (a subset of microfluidic devices) integrate almostall medical tests or diagnostic operations performed in a hospital on asingle microfluidic chip. The channels forming such microfluidicsdevices handle small fluid volumes and are connected together so as toachieve a desired function such as mixing of a sample, moving the samplethrough the device, reacting the sample with different reagents, and soon. These chips may be inserted into machines to control the performanceof a test and measure the results.

However, it has been found that handling a sample in a microfluidicsdevice can be very difficult. In such small channels as are found on aconventional LOC, it is difficult to apply external forces to move thesample from one site to another to perform different actions on thesample. There is also a limit to the complexity of a LOC device whichoperates purely using capillary action. Furthermore, owing to the smallsample sizes of LOC's, the devices have reduced sensitivity and theprobability of a target being present in the sample is thus reduced.

An alternative approach is to use a fluidic cartridge. The scale of thecomponents of a fluidic cartridge is larger than for a microfluidicdevice, and so it becomes possible to move a sample through variousdifferent sites to perform different actions on it. This makes itpossible to perform more complex tests than may be conducted usingtypical LOC devices, whilst still providing an analytical system ofpotential use in a local GP surgery.

Scientific assays useful in medical diagnostics have increasinglyinvolved biochemical procedures, such as the polymerase chain reaction(“PCR”). The PCR assay has provided a powerful method of assaying forthe presence of defined segments of nucleic acids. It is thereforedesirable to perform a PCR assay on a fluidic cartridge.

Reducing PCR to the microchip level is important for portable detectiontechnologies and high-throughput analytical systems. The method can beused to assay body fluids for the presence of nucleic acid specific forparticular pathogens, such as the Chlamydia trachomatis bacterium, HIVor any other pathogenic microbe.

The introduction of commercially available automated DNA amplificationassays has allowed more laboratories to introduce these technologies forroutine testing of specimens. However, there is a need to improve thefluidic devices used for this purpose.

With disposable fluidic cartridges adapted for use only once with acartridge reader, there exists a problem of ensuring that a usedcartridge is not reused by mistake. Such errors can be mitigated to someextent in a single laboratory which could make use of a central databasefor logging a serial number of each cartridge which is used, and thencross-checking a cartridge inserted into a laboratory machine againstthe central database. However, this solution is not scalable to accountfor decentralised diagnostic equipment in a large number of GPsurgeries, for example, around the country, and a simplified solution issought.

It is commonplace to provide simple mechanical arrangements forindicating to a machine the status of a readable medium insertedtherein. For example, video and tape cassettes comprise breakableplastic tabs which indicate to a video or tape cassette player, whetherthe cassette may be recorded onto. This solution is laborious for afluidic cartridge, however, and requires the addition of a separatemechanism taking up space and increasing the overall cost and complexityof the cartridge.

SUMMARY

Accordingly, in a first aspect, the present invention provides adisposable fluidic cartridge having a recess therein and amechanically-actuated mechanism disposed within the recess for effectingan operation of the fluidic cartridge; and further comprising: amachine-readable identification code on a breakable label covering atleast a portion of the recess such that the portion of the breakablelabel covering the recess may be broken upon insertion of a mechanicalactuator into the recess to actuate the mechanically-actuated mechanism,thereby rendering the identification code unreadable and preventingreuse of the cartridge.

Such a cartridge offers a simple yet reliable solution for ensuring thatit cannot be reused, even when there is no central database forrecording which cartridges have been used and which have not. If areader is configured only to begin the test if it is able to detect acode on a portion which is destroyed during use of the cartridge, thatcartridge can only ever be used once.

Preferably, the actuatable mechanism is a collapsible blister configuredto expel its contents into a fluid pathway within the fluidic cartridgeas it is collapsed. By providing a mechanism in the blister which isindispensable to the function of the cartridge, operation of thecartridge and destruction of the code are inextricably linked.

Preferably the collapsible blister contains a liquid component forconducting a diagnostic test. For example, the liquid component may beone of a reagent, a lysis buffer, a wash buffer and an elution buffer.

Preferably, the machine-readable identification code is a bar-code or aQR-code, which are straightforward to implement and have a high successrate for being read by a machine.

Preferably the machine-readable identification code takes up an area onbreakable label having a length and a width, and wherein at least onethe length and the width of said area is fully contained within theportion of the breakable label covering the recess. In other words, aportion of the code is completely destroyed along at least one dimensionbecause there is no part of that portion of the code which does notoverlie the recess.

Preferably the breakable label which covers the recess is separated fromthe remainder of the breakable label by a perforation to facilitatebreaking of the label. This ensures a complete breakage of the breakablelabel. Alternatively, the perforation may be provided as one or morelines crossing the recess.

In a second aspect, the present invention provides a cartridge readerfor receiving a disposable fluidic cartridge having a recess, amechanically-actuated mechanism disposed within the recess for effectingan operation of the fluidic cartridge, and a machine-readableidentification code on a breakable label covering at least a portion ofthe recess, the reader comprising: a sensor for detecting theidentification code of a disposable cartridge inserted into thecartridge reader; and a mechanical actuator configured extend into therecess to actuate the mechanically-actuated mechanism and break theportion of the breakable label covering the recess thereby rendering theidentification code unreadable and preventing reuse of the cartridge;wherein: the reader is configured only to extend the mechanical actuatorinto a recess of a cartridge inserted into the reader if the sensordetects an identification code on that cartridge.

Preferably the mechanical actuator is a foot configured to break theportion of the breakable label covering the recess and collapse acollapsible chamber located within the recess of the cartridge. Byproviding a reliable mechanism capable of collapsing the blister whichis indispensable to the function of the cartridge, operation of thecartridge and destruction of the code are inextricably linked.

Preferably the cartridge reader is configured to perform a diagnostictest on a sample in a disposable cartridge inserted into the reader.

Preferably the cartridge reader is configured to activate the sensor toattempt to detect an identification code upon insertion of a cartridgeinto the cartridge reader. This provides additional oversight byensuring the check is always carried out, and also makes the machineeasier to use.

In a third aspect, the present invention provides a method of using adisposable fluidic cartridge having a recess, a mechanically-actuatedmechanism disposed within the recess for effecting an operation of thefluidic cartridge, and a machine-readable identification code on abreakable label covering at least a portion of the recess, the methodcomprising: inserting the disposable cartridge into a cartridge reader;attempting to detect an identification code on the breakable label onthe disposable cartridge; and, if successful, extending a mechanicalactuator into the recess on the disposable cartridge to break theportion of the breakable label covering the recess, thereby renderingthe identification code unreadable, and actuate themechanically-actuated mechanism.

Preferably, the actuatable mechanism is a collapsible blister andwherein the step of actuating the actuatable mechanism comprisescollapsing the collapsible blister to expel its contents into a fluidpathway within the fluidic cartridge.

Preferably, the collapsible blister contains a liquid component forconducting a diagnostic test, the method further comprising the step ofperforming a diagnostic test on a sample in the disposable cartridge.

If the step of attempting to detect an identification code on thebreakable label is unsuccessful, the cartridge reader preferably rejectsthe cartridge.

The liquid component is preferably one of a reagent, a lysis buffer, awash buffer and an elution buffer.

The machine-readable identification code is preferably a bar-code or aQR-code, which are straightforward to implement and have a high successrate for being read by a machine.

Preferably, the step of attempting to detect an identification code istriggered upon insertion of the disposable cartridge into the cartridgereader. This provides additional oversight by ensuring the check isalways carried out, and also makes the machine easier to use.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of an exemplary fluidic cartridge in whichthe invention may be provided.

FIG. 2 is a top view of an exemplary fluidic cartridge in which theinvention may be provided.

FIG. 3 is an exploded view of the exemplary fluidic cartridge of FIG. 2.

FIG. 4 is a perspective view of the housing of the exemplary fluidiccartridge of FIG. 2.

FIG. 5 is a perspective view of the blister sub-assembly of theexemplary fluidic cartridge of FIG. 2.

FIG. 6A is a top view of the pneumatic layer of the exemplary fluidiccartridge of FIG. 2.

FIG. 6B is a bottom view of the pneumatic layer of the exemplary fluidiccartridge of FIG. 2.

FIG. 7 is a top view of the pneumatic foil of the exemplary fluidiccartridge of FIG. 2.

FIG. 8A is a top view of the fluidic layer of the exemplary fluidiccartridge of FIG. 2.

FIG. 8B is a bottom view of the fluidic layer of the exemplary fluidiccartridge of FIG. 2.

FIG. 9 is a top view of the fluidic foil of the exemplary fluidiccartridge of FIG. 2.

FIG. 10 is a top view of the electrode layer of the exemplary fluidiccartridge of FIG. 2.

FIG. 11 is a section view of an advantageous valve arrangement which mayform an isolated inventive aspect.

FIG. 12 is a section view of another advantageous valve arrangementwhich may form an isolated inventive aspect.

FIG. 13a is a section view of an advantageous inlet port arrangementwhich may form an isolated inventive aspect.

FIG. 13b is a perspective section view of the inlet port arrangement ofFIG. 13 a.

FIG. 14a is a section view of an advantageous capture column arrangementwhich may form an isolated inventive aspect.

FIG. 14b is a perspective section view of a portion of the capturecolumn arrangement of FIG. 14 a.

FIG. 15a is a section view of an advantageous waste chamber arrangementwhich may form an isolated inventive aspect.

FIG. 15b is a perspective section view of the waste chamber arrangementof FIG. 15 a.

FIG. 16 is a top view of an exemplary cartridge with which the presentinvention may be used.

FIG. 17 is a top view of a cartridge according to the present invention.

DETAILED DESCRIPTION

Embodiments of the invention will now be described in the context of anexemplary fluid cartridge in which the invention is implemented. Whilstnot necessary to understand the present invention, it is beneficial toprovide general description of the principles of the structure,manufacture, function and use of the fluidic cartridge and associatedmethods for performing a test.

The exemplary fluidic cartridge and associated methods chosen toillustrate the present invention are for the detection of Chlamydiatrachomatis bacterium using PCR amplification and electrochemicaldetection. However, the skilled person would understand that theinvention is not limited to the exemplary fluidic cartridge andassociated methods, and is suitable for use in with various differentcartridges for a wide variety of sample analysis techniques orbiological assays; for example, assays of target nucleic acid sequencesin a liquid sample.

Those skilled in the art will understand that the devices and methods ofthe invention described herein and illustrated in the accompanyingdrawings are non-limiting exemplary embodiments and that the scope ofthe present invention is defined solely by the claims. The featuresillustrated or described in connection with one exemplary embodiment maybe combined with features of other embodiments. Such modifications andvariations are included within the scope of the present disclosures.

The exemplary cartridge comprises: a fluidic portion through which thesample flows and in which nucleic acid amplification and detection takeplace; a pneumatic portion which controls flow through the fluidicportion; and at least two electrodes which provide a potentialdifference for the detection of an amplified nucleic acid of interest.The fluidic portion and pneumatic portion may be constructed of afluidic layer, a fluidic foil, a pneumatic layer and a pneumatic foilsuch as those described in relation to the exemplary cartridge below.However, the fluidic portion does not necessarily consist only of afluidic layer and a fluidic foil and the pneumatic portion does notnecessarily consist only of a pneumatic layer and a pneumatic foil.Rather, the layers may interact to produce the fluidic portion and thepneumatic portion such that parts of all or some of the layers make upeach portion. Rather than referring to the particular layers of thecartridge, the fluidic portion refers to the particular areas of thecartridge which provide the function of allowing controlled sample flow,and the pneumatic portion refers to the particular areas of thecartridge which provide the function of controlling the flow through thefluidic portion.

The housing, fluidic portion and pneumatic portion are made of plastic.By plastic is meant a synthetic or natural organic material that may beshaped when soft and then hardened, including resins, resinoids,polymers, cellulose derivatives, casein materials, and protein plastics.Examples of plastics from which the cartridge may be constructedinclude, but are not limited to thermoplastics, for examplepolycarbonate, polyethylene terephthalate, cyclic olefin copolymers suchas Topaz, acrylonitrile butadiene styrene, and thermoplastic elastomers,for example polypropylene. Plastic housings, fluidic portions andpneumatic portions can include components which are not made of plastic(e.g. blisters made from metal foil, metallic inserts at the sampleinlet), but they are formed primarily from plastic. The use of plasticmaterials facilitates economical manufacture of the cartridges.

Whilst the pneumatic and fluidic foils may be made from a metal foil,the preferred materials are plastic including those mentioned above. Inparticular, it is preferred that foils are a polyethyleneterephthalate/polypropylene composite.

The target nucleic acid sequence is any nucleic acid to be detected in asample. The target nucleic acid(s) to be amplified and detected in thecartridge will usually be DNA, but it is also possible to amplify anddetect RNA. In some embodiments a cartridge may permit amplificationand/or detection of both DNA and RNA targets.

The liquid sample is the composition which is introduced into thecartridge in order to determine whether the target nucleic acid(s) ofinterest is/are present. The sample may be a composition in which thenucleic acid to be detected is suspected to be present (e.g. forclinical diagnosis), or may be a composition in which the nucleic acidto be detected is potentially present (e.g. for contamination testing).

The liquid sample can have various sources. For instance, it can bematerial obtained from an animal or plant (e.g. for diagnosis ofinfections or for genotyping). Such samples may be obtained with minimalinvasiveness or non-invasively, e.g., the sample may be obtained from ananimal using a swab, or may be a bodily fluid. As an alternative, thesample may be material obtained from food or water (e.g. forcontamination testing). The sample will usually include cells, and thetarget nucleic acid (if present) can be extracted from these cellswithin the cartridge. One skilled in the art will appreciate thatsamples can be diluted or otherwise treated prior to being introducedinto the cartridge, but it is preferred that the cartridge can handlematerial which has not been pre-treated in this way.

An animal from whom the sample is obtained may be a vertebrate ornon-vertebrate animal. Vertebrate animals may be mammals. Examples ofmammals include but are not limited to mouse, rat, pig, dog, cat,rabbit, primates or the like. The animal may be a primate, and ispreferably a human. Thus the cartridge can be used for clinicaldiagnosis of human samples.

In addition to analysing a sample, the cartridge can analyse a positiveand/or negative control to provide confirmation that the cartridge isfunctioning as expected. The control(s) can be introduced into thecartridge by a user, or can be included within a cartridge before use.

The inclusion of an internal positive control nucleic acid allows a userto identify whether a negative result for the sample has been obtainedbecause the nucleic acid amplification has been unsuccessful (falsenegative). If the positive control nucleic acid fails to be detected inthe detection chamber, despite its presence in an amplification chamber,the user will be able to identify the test as a potential false negativeresult, and can perform another test.

The inclusion of an internal negative control allows a user to identifywhether a positive result has been falsely obtained because of thepresence of contamination. A negative control can involve performing PCRin a chamber in which no nucleic acid is provided, or in which a sampleundergoes an amplification reaction without necessary components e.g.PCR without primers. If nucleic acid is nevertheless detected in thedetection chamber, despite its intended absence in an amplificationchamber, the user will be able to identify the test as a potential falsepositive result, and can perform another test.

A positive control nucleic acid may be any nucleic acid that will not befound in a sample used in the cartridge. The internal control DNA may betaken from a bacterium that is not pathogenic to animals and whichcontains a nucleic acid that is highly specific to the bacterium. Oneexample of a possible bacterium from which the control nucleic acid maybe taken for an animal sample is Pectobacterium atrosepticum, althoughany control nucleic acid may be used that will not be present in asample.

The fluidic portion of the cartridge comprises channels and chambersthrough which sample flows. The flow of sample through the cartridge iscontrolled in two ways. Firstly, the fluidic portion has a gas inlet.The gas inlet is connected to a gas supply, and injection of gas intothe fluidic portion via this inlet allows the sample to be pusheddownstream through the cartridge, towards the detection chamber. The gassupply may be provided by the reader. As an alternative, the gas supplymay be an on-board gas supply. Preferably, the gas supply is provided byan external source and the gas inlet is connected to a pneumatic circuitsuch that the gas supply is provided via a pneumatic inlet on thecartridge. Secondly, at least one pneumatically controlled valvecontrols local movement of the sample through the fluidic portion. Thepneumatically controlled valve(s) may be controlled independently ofother pneumatically controlled valves and may be controlledindependently of the gas supply that generally causes downstreammovement of the sample via the gas inlet. The gas inlet and thepneumatically controlled valve(s) also permit sample to be flushedthrough the fluidic portion e.g. to exclude excess volumes of material.The fluidic portion also has an exhaust which allows air and wastematerial to exit the channels and chambers of the fluidic portionwithout a build-up of pressure occurring in the cartridge. Preferably,the exhaust comprises a waste chamber and/or a waste vent.

The fluidic portion of the cartridge includes reagents and/or physicalcomponents for cell lysis and nucleic acid separation. These may be anyreagents or physical components that are capable of lysing cells andseparating nucleic acids from cell debris and other cellular components.For instance, they may comprise (i) a lysis buffer which is capable ofcausing lysis of target cells which may be present in the sample e.g.buffers including a detergent such as nonyl phenoxypolyethoxylethanol(available as NP-40) or t-octylphenoxypolyethoxyethanol, (available asTriton X 100), or including guanidine thiocyanate, and/or (ii) a capturesupport or column which specifically binds nucleic acids but does notbind other undesired cellular components (e.g. proteins and lipids). Thecapture column comprises a capture filter and may additionally comprisea depth filter. The filters may be made of glass fibres (available asWhatman filters), or may be made of silica, although any column orsupport which is capable of separating nucleic acids from other cellularcomponents may be used. Elution using a wash buffer to remove celldebris and other cellular components, followed by elution using anelution buffer to elute the separated nucleic acids from the capturesupport or column can be undertaken such that the capture column canseparate nucleic acids from cell debris and other cellular components.

A channel through which the sample flows fluidly connects the sampleinlet to at least one amplification chamber where nucleic acidamplification can take place. The purpose of the amplificationchamber(s) is to permit amplification of any target nucleic acid ofinterest that is present in the sample (and, where present, any positivecontrol nucleic acid). Any nucleic acid amplification method may be usedand these are described in more detail below in relation to an exemplarycartridge. The different nucleic acid amplification reagents that arerequired for different nucleic acid amplification methods are well knownin the art. These reagents are provided in or upstream of theamplification chamber(s) such that the sample (and any positive control)includes all necessary reagents for nucleic acid amplification once itreaches the amplification chamber. Adaptation of a nucleic acidamplification method according to the target nucleic acid to be detectedis also well known in the art (e.g. design of primers). The skilledperson would therefore be able to adapt the reagents for nucleic acidamplification accordingly. The term “chamber” does not denote anyparticular size or geometry, but instead it means a region within thefluidic portion which is designed to permit nucleic acid amplificationto occur. Thus, for instance, it could be a region in which the samplecan be fluidically isolated (e.g. via the use of pneumaticallycontrolled valves) while the steps required for nucleic acidamplification (e.g. thermocycling, etc.) occur, and it can be locatedwithin the cartridge so that it is in the proximity of any externalresources that are needed (e.g. next to a heat source within a cartridgereader, thereby permitting thermal cycling to occur).

Multiple test amplification channels and/or chambers may be included inthe cartridge. The different test amplification channels and/or chambersmay include reagents required to amplify different nucleic acids ofinterest. Therefore using multiple amplification test channels and/orchambers allows multiple tests to be performed on a single cartridge,simultaneously (including any controls). As an alternative, reagents foramplification of multiple different nucleic acids may be present in asingle amplification chamber, and the different nucleic acids (whethermultiple target nucleic acids, or a target nucleic acid and a controlnucleic acid) may be amplified simultaneously in the same amplificationchamber.

A further channel through which the sample flows after nucleic acidamplification fluidly connects the at least one amplification chamber toat least one detection chamber where the results of nucleic acidamplification can be detected. In or upstream of the detection chamberare reagents for nucleic acid detection such that the sample includesall necessary reagents for the detection once it reaches the detectionchamber. The reagents for nucleic acid detection may be specific for theparticular target nucleic acid, i.e. they may allow for detection of thepresence of the specific nucleic acid sequence. As an alternative, thereagents for nucleic acid detection may be generic reagents to detectthe presence of any nucleic acids. Such generic reagents may be used ifall nucleic acids other than the target nucleic acid are removed priorto detection. For example, this may be achieved by providing a nucleasethat is capable of hydrolysing all nucleic acids present in the sampleother than the target nucleic. The amplified target nucleic acid can beprotected from hydrolysis, for example by inclusion of chemicalmodifications in the primers which are incorporated into the amplifiedproduct and which cannot be hydrolysed. Reagents for nucleic aciddetection are described below in relation to an exemplary cartridge butusually comprise a probe including a label. The probe is capable ofhybridising to the amplified nucleic acid which has been amplified inthe amplification chamber(s). Following hybridisation of the probe tothe amplified nucleic acid, the detection of the nucleic acid may occurvia a detectable change in the signal from the label. In someembodiments the change may be caused by hydrolysis of the probe. Wherethe probe is hydrolysed, hydrolysis is usually achieved using a doublestrand specific nuclease, which can be an exonuclease or anendonuclease. Preferably, the nuclease is T7 endonuclease. The signalfrom the label is capable of undergoing a change following hydrolysis ofthe probe. This is due to a change in the environment of the label whenit moves from being bound to the rest of the probe to being free fromthe rest of the probe or bound to a single nucleotide or a short part ofthe probe. Further details of the types of probes and detection methodsthat may be used can be found in Hillier et al. Bioelectrochemistry, 63(2004), 307-310. As an alternative, methods for causing a detectablechange in the signal from the label which do not rely on hydrolysis ofthe probe may be used e.g. see Ihara et al. Nucleic Acids Research,1996, Vol. 24, No. 21 4273-4280. This change in environment of the labelleads to a change in the signal from the label. The change in signalfrom the label can be detected in order to detect the presence of thenucleic acid of interest.

Where a positive control nucleic acid is used, the reagents for nucleicacid detection will additionally include a positive control probeincluding a label. The positive control probe is capable of hybridisingto the amplified control nucleic acid. The signal provided by the labelsof the positive control and target probes may be the same, but presentin separate detection chambers such that the signals corresponding tothe control and test nucleic acids can be distinguished. As analternative, the signal provided by the labels of the control and targetprobes may be different, such that the signals are distinguishable fromone another, even if the probes are present in the same detectionchamber.

Multiple test detection channels and/or chambers may be included in thecartridge. The different test detection channels and/or chambers mayinclude reagents required to detect different nucleic acids of interest.Therefore using multiple detection test channels and/or chambers allowsmultiple tests to be performed on a single cartridge, simultaneously. Asan alternative, reagents for detection of multiple different nucleicacids may be present in a single detection chamber, and the differentnucleic acids (whether multiple target nucleic acids or a target nucleicacid and a control nucleic acid) may be detected simultaneously in thesame detection chamber.

The label is detectable by use of the cartridge's electrodes, and so thelabel will usually be an electrochemical label, such as a ferrocene.Examples of labels which may be used can be found in WO03/074731,WO2012/085591 and PCT/GB2013/051643. Signal emitted by the label can bedetected by a cartridge reader.

The pneumatic portion of the cartridge comprises at least one pneumaticcircuit which each control at least one pneumatically controlled valve.The pneumatic portion controls sample flow through the cartridge by theopening and closing of pneumatically controlled valves. The opening andclosing of the valves is controlled by changes in pneumatic pressure inthe pneumatic circuit that is applied through a pneumatic pressureinlet. Usually, the cartridge contains many pneumatically controlledvalves. The pneumatically controlled valves may be controlled byseparate pneumatic pressure inlets. These valves can be used to preventdownstream movement of sample through the fluidic portion untilnecessary steps have been performed and/or to prevent unwanted reversemovement of sample upstream. For example, a valve may be providedupstream of the at least one amplification chamber in order to preventdownstream movement into the at least one amplification chamber untilcell lysis and nucleic acid separation has taken place. Following celllysis and nucleic acid separation the valve upstream of the at least oneamplification chamber may be opened in order to allow downstream flow.It can then be closed again, to prevent backflow out of the chamber backtowards the sample inlet.

The cartridge comprises at least two electrodes which can provide apotential difference across the at least one detection chamber. Thepotential difference causes current to flow through the at least onedetection chamber, thereby permitting the detection of signal fromelectrochemically active labels.

An exemplary cartridge which operates according to the above descriptionwill now be described with reference to the accompanying drawings.

1. The Exemplary Cartridge 1.1 Overview

The exemplary cartridge described below is intended to be a single-use,disposable cartridge for performing a test on a sample introduced intothe cartridge. The exemplary cartridge is a fluidic cartridge withchannels of an appropriate scale (as detailed hereafter). However, theinvention may be performed on a microfluidic device, or an LOC. Once thetest has been run, it is preferred that the cartridge is disposed of.However, if desired, the cartridge may be sent for re-processing toenable it to be used again.

It is preferred that the cartridge comprises all of the biologicalagents necessary for conducting the test of choice. For example, theexemplary cartridge is used for detecting the presence, absence oramount of a pathogen of interest. Any pathogen may be detected. Examplesof pathogens which may be detected by the cartridge are Chlamydiatrachomatis, Trichomonas vaginalis, Neisseria gonorrhoea, Mycoplasmagenitalium and methicillin resistant Staphylococcus aureus. To that endthe cartridge comprises reagents for nucleic acid amplification. Nucleicacid amplification may be performed using any nucleic acid amplificationmethod. The nucleic acid amplification method may be a thermocyclingmethod in which the temperature at which the method is performed isvaried such that different steps of the amplification are able to takeplace at different temperatures within the cycle. For example melting,annealing of primers and extension may each be performed at differenttemperatures. By cycling through the temperatures, the timing of each ofthe steps of the method can be controlled. As an alternative, thenucleic acid amplification may be an isothermal method in which thetemperature is kept constant. In both the thermocycling and theisothermal nucleic acid amplification methods, the temperature iscontrolled during nucleic acid amplification.

Examples of nucleic acid amplification methods are the polymerase chainreaction (PCR), the ligase chain reaction (LCR), strand displacementamplification (SDA), transcription mediated amplification, nucleic acidsequence-based amplification (NASBA), helicase-dependent amplificationand loop-mediated isothermal amplification. The reagents for nucleicacid amplification will vary depending of the nucleic acid amplificationmethod used but include a polymerase and nucleotide triphosphates.

As explained below, the cartridge also comprises detection reagentswhich are capable of detecting the presence or absence of amplifiednucleic acids which are the product of the nucleic acid amplificationmethod. The reagents for nucleic acid detection comprise a probe whichis capable of hybridising to the amplified nucleic acid. The probeincludes a ferrocene label. Following hybridisation of the probe to theamplified nucleic acid, the detection of the nucleic acid occurs via adetectable change in the signal from the label. The change is caused byhydrolysis of the probe, which is achieved using a double strandspecific nuclease. The nuclease is a T7 endonuclease. The ferrocenegives different electrochemical signals when it is part of a probe orwhen it is attached only to a single nucleotide, and so hydrolysis iseasily detected. Thus, the change in signal from the label permitsdetection of the presence of the nucleic acid of interest.

The electrodes allow the detectable change in the signal from the label,which occurs in the presence of the target nucleic acid, to be detected.

The cartridge is configured for use with a cartridge reader (not shown).The cartridge comprises a number of pneumatic, mechanical, thermal andelectrical interfaces (described in more detail below) through which thereader interacts with the cartridge to perform the test. Hence, in use,the cartridge would be inserted into the reader, and the reader would beactivated to begin interacting with the cartridge via the interfaces toperform the test. For the purposes of understanding the presentinvention, it is not necessary to describe exactly how the cartridgeinteracts with the reader to conduct a particular test and provide thetest results, but an overview of an exemplary operation of a cartridgeis provided hereafter.

1.2 Schematic Diagram of the Exemplary Cartridge

Before explaining the structure and arrangement of the components of anexemplary fluid cartridge in detail, it is helpful to describe thelayout of the exemplary cartridge at a high level with reference to theschematic shown in FIG. 1.

It is convenient to consider the overall layout of the cartridge interms of the flow of liquids, including the liquid sample, through thecartridge. Unless otherwise specified hereafter, the passage of liquidsincluding the liquid sample and the liquid buffers is referred to as the‘fluid pathway’ which has an upstream end and a downstream end. Unlessotherwise specified hereafter, ‘downstream’ generally refers to thedirection of flow of the liquids and ‘upstream’ refers to the directionopposite the direction of flow. The fluid pathway in the exemplarycartridge may have different branches (and thus form different fluidpathways), but all pathways have a recognisable direction of flow whichpermit a skilled person to identify the upstream and downstreamdirections. However, there is an exception to this general definition,which is when the liquid sample is pumped between the mixing chamber 10and the bellows 20. In this case, fluid is intermittently pumped backupstream in the opposite direction to its general direction of fluidflow, which is downstream. This mixing serves to mix the lysis andsample and to rehydrate the internal control.

The liquid sample is introduced into the cartridge at a sample mixingchamber 10 through an entry port. A particular arrangement of apreferred entry port may itself form an isolated inventive aspect of thecartridge, as described further in section 3, below. A sample indicator12 is fluidly coupled to the sample mixing chamber 10 such that a sampleintroduced into the sample mixing chamber 10 is visible in the sampleindicator 12. Also connected to the sample mixing chamber 10 is ablister 14 containing a lysis buffer. The lysis buffer comprisesguanidine thocyanate. Once the sample has been introduced into thesample mixing chamber 10, and a test is started, the lysis blister 14 iscollapsed so as to expel the lysis buffer into the sample mixing chamber10 where it mixes with the liquid sample introduced therein.

Downstream of the sample mixing chamber 10, along a main channel 16, isa coarse filter 18. The coarse filter 18 filters out any large debris inthe liquid sample, such as skin or bodily hair, as the liquid samplepasses through main channel 16.

Downstream of the coarse filter 18, along the main channel 16, is abellows 20 having an upstream bellows valve 22 a and a downstreambellows valve 22 b. As described in more detail below, the bellows 20,together with its upstream and downstream valves 22 a-b, is capable ofpumping the liquid sample from the upstream end of the fluid pathway(i.e. from the sample mixing chamber 10) to the downstream end. Insummary, this is achieved by virtue of flexible membranes within thebellows 20 and the upstream and downstream bellows valves 22 a-b whichactuate to create local pressure differentials to, on the one hand, drawin the liquid sample from the sample mixing chamber 10 into the bellows20 and, on the other hand, from the bellows 20 further downstreamthrough the main channel 16. This is achieved by carefully choreographedpneumatic actuation of the flexible membranes in the valves. Particulararrangements of a preferred valve may themselves form isolated inventiveaspects of the cartridge, as described further in section 3, below.

Downstream of the bellows along the main channel 16 is a capture column24. The purpose of the capture column 24 is to separate nucleic acidsfrom cell debris and other cellular components. The capture columncomprises a capture filter and a depth filter both made of glass fibres.A particular arrangement of a preferred capture column may itself forman isolated inventive aspect of the cartridge, as described further insection 3, below.

Two branch channels 26, 28 join the main channel 16 between thedownstream bellows valve 22b and the capture column 24. The purpose ofthe branch channels is to introduce liquid buffers necessary forperforming the desired test. For example, with the test conducted by theexemplary cartridge, it is necessary to introduce an elution buffer anda wash buffer into the main channel once the sample has passed through.The wash buffer is contained in a wash buffer blister 30 and the elutionbuffer is contained in an elution buffer blister 32. The introduction ofthe wash buffer and elution buffer into the main channel 16 iscontrolled by wash buffer valve 34 and elution buffer valve 36,respectively. At the appropriate point in the test, the wash and elutionbuffer blisters 30, 32 are collapsed so as to expel the wash and elutionbuffers into the branch channels 26, 28 and thence into the main channel16 through the wash and elution buffer valves 34, 36.

Downstream of the capture column 24, along a waste branch 16 a of themain channel 16, is a waste chamber 38. A particular arrangement of apreferred waste chamber may itself form an isolated inventive aspect ofthe cartridge, as described further in section 3, below. The purpose ofthe waste chamber 38 is to collect the cell debris and cellularcomponents other than nucleic acids and contain them, thereby preventingthem from entering the test channel 54 a or the control channel 54 b.The waste chamber 38 is vented to atmosphere through a waste vent 40,and an aerosol impactor 42 is provided between the waste chamber 38 andthe waste vent 40 to prevent particulate matter from escaping from thewaste chamber 38 into the atmosphere. A waste chamber valve 44 in themain channel waste branch 16 a of the main channel 16 permits andprevents fluids passing into the waste chamber 38 at appropriate pointsduring the test.

Downstream of the capture column 24, along an elution branch 16 b of themain channel 16, is an elution chamber 46. The purpose of the elutionchamber 46 is to allow the sample preparation to settle and for bubblesto disperse before the sample enters the amplification chambers. Anelution chamber valve 48 in the elution branch 16 b of the main channel16 permits and prevents fluids passing into the elution chamber 46 atappropriate points during the test.

Downstream of the elution chamber 46 is a convoluted mixing channel 52.Here the prepared sample is mixed prior to passing through the isolationvalve 50.

In the present application, the components upstream of the isolationvalve 50 are referred to as being comprised in the ‘front end’ of thecartridge, whilst the components downstream of the isolation valve 50are referred to as being comprised in the ‘back end’ of the cartridge.Broadly speaking, the liquid sample is prepared for analysing in thefront end of the cartridge, and the analysis is carried out on thesample in the back end of the cartridge.

The isolation valve 50 is open to permit the prepared liquid sample topass from the front end to the back end of the cartridge. At anappropriate point in the test, after the liquid sample has been preparedand is within the back end of the cartridge for analysis, the isolationvalve 50 is closed to prevent any of the sample from re-entering thefront end. Once the isolation valve 50 is closed, it cannot be openedagain. The isolation valve 50 also acts as a safeguard in case of apower failure, wherein the reader closes the isolation valve 50 toprevent leakage.

Downstream of the isolation valve 50, the fluid pathway splits into anamplification test channel 54 a and an amplification control channel 54b. Each of the amplification channels 54 a-b comprises an amplificationchamber 56 a-b having an amplification chamber inlet valve 58 a-b and anamplification chamber outlet valve 60 a-b. Any nucleic acidamplification method may be performed in the nucleic acid amplificationchamber. If PCR is used, the nucleic acid amplification chambers containa thermostable DNA polymerase, dNTPs, a pair of primers which arecapable of hybridising to the nucleic acid to be amplified. Optionally,the nucleic acid amplification chambers may additionally contain buffersalts, MgCl₂, passivation agents, uracil N-glycosylase and dUTP. Anexample of a thermostable DNA polymerase that may be used is Taqpolymerase from Thermus aquaticus.

Each of the nucleic acid amplification chambers in the exemplarycartridge comprises reagent containment features in the form of firstand second shallow wells formed in the fluidic layer. The reagents to beused in the cartridge are spotted in the wells. In the exemplarycartridge, the test-specific reagents and the generic reagents areisolated from each other by spotting each in a different well. Hence,the test-specific reagents are spotted in a first well in the chamberand the generic reagents are spotted in a second well in the chamber. Byspotting the reagents separately, it is easier to swap the test-specificreagents during manufacture for a different set of test-specificreagents, so as to perform a different test, whilst keeping the genericreagents as they are.

In the exemplary cartridge, the ratio of nucleic acid amplificationchambers to detection chambers is 1:2. The prepared sample enters theback end of the cartridge at the isolation valve 50 and is split intotwo nucleic acid amplification chambers. After processing, the each ofthe two processed measures of sample from the nucleic acid amplificationchamber is split into two detection chambers. Therefore, for each sampleintroduced into the exemplary cartridge, four detection chambers may befilled from two nucleic acid amplification chambers, thus facilitatingduplex amplification and 4-plex detection.

However, it will be appreciated that one or three or more nucleic acidamplification chambers may be provided to provide any level ofmultiplexing desired, and that the number of the detection chambersprovided may be adjusted accordingly to maintain a 1:2 ratio of nucleicacid amplification chambers to detection chambers.

The ratio 1:2 is preferred for the exemplary cartridge because such aratio allows twice the number of target nucleic acids to be assayedcompared to the number of different labels required for detection in thedetection chambers. However, it will be appreciated that the ratio maybe changed depending on the number of labels and PCR targets for theliquid sample. For instance, the ratio may be 1:1, 1:3 or 1:n such thatthere are n detection chambers branching from the main channel of eachfluid pathway when there are n times as many multiplexed PCR targets forthe number of labels.

PCR primers specific for Chlamydia trachomatis are dried down in theamplification chamber in the amplification test channel together withthe other reagents required for nucleic acid amplification. PCR primersspecific for a positive control nucleic acid are dried down in theamplification chamber in the amplification control channel together withthe other reagents required for nucleic acid amplification. A positivecontrol nucleic acid is also provided in the amplification chamber inthe amplification control channel, taken from Pectobacteriumatrosepticum. The dried down reagents are reconstituted when the liquidsample reaches them.

Downstream of the amplification chamber outlet valves 60 a-b each of theamplification channels 54 a-b splits into two further detectionchannels, leading to two detection chambers for each amplificationchamber, giving a total of four detection chambers 62 a-d in total. Thereagents for nucleic acid detection, including the target probe, aredried down in the detection chambers 62 a-d downstream of the testamplification chamber 56 a or 56 b. The reagents for nucleic aciddetection including the control probe are dried down in the detectionchambers downstream of the control amplification chamber 56 a or 56 b(whichever is not the test chamber mentioned above). Each detectionchamber 62 a-d is provided with its own gas spring 64 a-d which forms adead end at the downstream end of the fluid pathway.

Reagents for nucleic acid detection are provided in detection chambers.The reagents for nucleic acid detection include probes having aferrocene label. These probes are capable of hybridising to theamplified nucleic acids. Following hybridisation of the probes to theamplified nucleic acids, the probes are hydrolysed by a double strandspecific nuclease¹ which causes the label to be freed from the rest ofthe probe. As explained above, freeing of the label from the rest of theprobe causes a detectable change in the signal from the label. Thecontrol probe is provided in separate detection chambers to the targetprobe and detection of the target nucleic acid and the control nucleicacid take place in different detection chambers, such that the signalsare distinguishable from one another.

Downstream of the amplification outlet valves 60 a-b, but upstream ofthe forks creating the four detection channels, two bypass channels 66a-b respectively join the two amplification channels 54 a-b. The purposeof the bypass channels 66 a-b is to remove excess liquid sample withinthe amplification channels 54 a-b before the liquid sample enters thedetection chambers 62 a-d. The bypass channels 66 a-b connect to abypass valve 68, which is also fluidly coupled to the elution chamberbranch 16 b of the main channel 16, downstream of the isolation valve50, before the channel splits into amplification channels 54 a and 54 b.

A particular arrangement of a preferred chamber in the cartridge, suchas the first and second amplification chambers or the first to fourthdetection chambers, may itself form an isolated inventive aspect of thecartridge, as described further in section 3, below.

It will be appreciated that the number of amplification chambers, andthe number of detection chambers in the exemplary cartridge may varydepending on the preferred implementation. Moreover, otherconfigurations of channels, chambers, valves and so on are possiblewithout departing from the scope of the invention, as defined by theclaims.

The physical structure and operation of the various components of theexemplary cartridge introduced above will now be explained withreference to FIGS. 2 to 10.

1.3 Physical Structure of an Exemplary Cartridge 1.3.1 Overview andExternal Features of the Exemplary Cartridge

An exemplary cartridge is shown in FIG. 2. As described above, thereader interacts with the cartridge through a plurality of interfaces.The interfaces shown in the exemplary cartridge 100 are: a pneumaticinterface 101; an electrical interface 102; a bypass valve interface103; and an isolation valve interface 104. Each of these interfaces isdescribed in more detail below. It will be appreciated that more orfewer interfaces could be provided, depending on the preferredimplementation.

Also provided in the cartridge, but not shown, is a thermal interface.The thermal interface allows the temperature of the amplificationchambers to be regulated to allow nucleic acid amplification to takeplace.

The exemplary cartridge 100 shown in FIG. 2 comprises an insertion end105 for insertion into the reader, and a non-insertion end 106.Proximate the non-insertion end 106 is a sample inlet 107 forintroducing a sample into the sample mixing chamber 10. In the exemplarycartridge, the sample will usually include cells, and the target nucleicacid (if present) can be extracted from these cells, but other fluidsamples such as swab eluate, urine, semen, blood, saliva, stool sweatand tears could be used in other implementations. The sample may beintroduced into the sample mixing chamber 10 through the sample inlet107 using a pipette, for example.

The exemplary cartridge 100 and reader are configured such that when thecartridge is inserted into the reader, all of the aforementionedinterfaces are actuatable by the reader. On the other hand, the sampleinlet 107 remains external to the reader such that a sample may beintroduced into the sample mixing chamber 10 whilst the cartridge isinserted into the reader.

The exemplary cartridge 100 shown in FIG. 2 further comprises a sampleindicator window 109, through which the sample indicator 12 is visibleto determine whether a sample has been introduced into the sample mixingchamber 10.

All of the pneumatic, mechanical and electrical interfaces in theexemplary cartridge 100 are located on the same face of the cartridge,in this case the top face 110. The thermal interface (not shown) isprovided on the bottom face of the cartridge. This simplifies the designof the reader, which may this provide the associated pneumatic,mechanical and electrical parts which interact with those interfaces inthe same region of the reader, thereby making best use of space. It alsoenables the thermal part of the reader to be provided away from thepneumatic, mechanical and electrical parts.

1.3.2 Internal Components of Cartridge

The exemplary cartridge 100 shown in FIG. 2 is formed from variouscomponents which shall now be described. FIG. 3 shows an exploded viewof the exemplary cartridge 100 of FIG. 2. The cartridge 100 comprises,from top to bottom, a housing 111, a blister sub-assembly 112, apneumatic foil 113, a pneumatic layer 114, a fluid layer 115 and afluidic foil 116. Also shown in FIG. 3 is an electrode layer 117, twofilters 118 and a plurality of absorbent pads 119, which will bedescribed in more detail below.

The housing 111 is manufactured from acrylonitrile butadiene styrene.The pneumatic and fluidic foils 113, 116 are manufactured from apolyethylene terephthalate/polypropylene composite. The pneumatic andfluidic layers 114, 115 are manufacture from polypropylene.

With the exception of the housing 111, filters 118 and pads 119, each ofthe components mentioned in the previous paragraph is adhered to itsadjacent component or components. Hence, the blister sub-assembly 112 isadhered to the pneumatic foil 113, which is adhered to the pneumaticlayer 114, which is adhered to the fluidic layer 115, which is adheredto the fluidic foil 116. The electrode layer 117 is adhered to fluidiclayer 115 also.

The adhesion of the layers to each other provides a series offluid-tight channels in the cartridge, together with associatedchambers, valves, pumps, bellows and other components. The channelspassing a liquid sample therethrough are liquid-tight and the channelspassing a gas therethrough are gas-tight. Optionally, all components areboth liquid tight and gas-tight. For example, recesses and openingsformed in one or both sides of the pneumatic and fluidic layers create,when sandwiched together and adhered to the pneumatic and fluidic foils,respectively, the shapes necessary to provide the aforesaid channels,chambers, valves, pumps, bellows and other components.

Each of the components referred to above in FIG. 3 will now be describedin more detail.

1.3.3 Housing 111

FIG. 4 shows housing 111 in more detail. As shown, housing 111 comprisesa generally rectangular upper surface 120 and walls 121 dependingtherefrom on all four sides (two of which are visible in FIG. 4). Aprincipal purpose of the housing 111 is to protect certain components ofthe cartridge, most notably the blister sub-assembly 112 and theisolation valve interface 104. It will therefore be noted that thehousing 111 is shorter than the pneumatic and fluidic layers 114, 115such that it overlies only a portion of those layers when the cartridge100 is assembled. In the exemplary cartridge 100, the pneumaticinterface 101, electronic interface 102, and bypass valve interface 103are not covered by the housing 111 to provide ease of access by thereader.

The upper surface 120 of the housing 111 has three apertures 122 a-ctherein, each having walls depending from the peripheries of theapertures to form, when the cartridge is assembled, three recesses. Thepurpose of the recesses is to house the blisters of the blistersub-assembly 112 such that the blisters may be accessed and pressed bythe reader, but are otherwise protected from accidental impact.Naturally, since the exemplary cartridge comprises three blisters, thehousing 111 comprises three corresponding apertures 122 a-c formingthree corresponding recesses. It will be appreciated that more or fewerblisters, apertures and recesses may be provided, depending on thepreferred implementation. Alternatively, the housing 111 could comprisea single aperture forming a single recess housing all availableblisters.

The side walls 121 of the housing 111 which run along the length of thehousing 111 between the insertion end 105 and the non-insertion end 106of the cartridge 100 comprise flanges 123 along at least a portion oftheir lower edges. The purpose of the flanges 123 is two-fold. Firstly,they comprise one or more windows 124 a-b for receiving a correspondingnumber of tabs formed in the pneumatic layer 114 to hold the cartridge100 together. Secondly, the flanges 123 are dimensioned so as toprotrude beyond the lower surface of the fluidic foil 116 when thecartridge is assembled, such that the fluidic foil 116 is suspendedabove a flat surface on which the cartridge 100 is placed. This preventsaccidental damage to the fluidic foil 116 which could otherwise result.

Although in the exemplary cartridge depicted in FIG. 4 flanges 123 areprovided along substantially the length of two opposing sides of thecartridge, it will be appreciated that flanges may be provided alongthree or four edges of the cartridge and still suspend the foil above aflat surface on which the cartridge is placed. Similarly, although thecartridge depicted in FIG. 4 shows flanges 123 extending alongsubstantially the entire length of the edge, a flange which extends onlypartially along an edge may be provided, or multiple flanges may beprovided along each edge.

The housing 111 further comprises, at the non-insertion end 106, a grip125 to facilitate insertion of the cartridge into and removal of thecartridge 100 from the reader by hand. The grip 125 comprises a seriesof ridges and grooves formed in the housing 111, but alternativestructures to increase friction, such as knurls, are also possible.

The housing 111 further comprises a sample inlet aperture 126 throughwhich a sample may be introduced into the sample mixing chamber 10 ofthe cartridge 100 using a pipette, for example. Surrounding the inletaperture 126 for a given diameter is a basin 127 recessed into the uppersurface 120 of the housing 111 to accommodate a certain amount ofspillage of the liquid sample. Whilst the basin 127 of the exemplaryembodiment is substantially flat, it may be sloped toward the inletaperture 126, such that any spillage drains through the inlet aperture126.

The exemplary housing 111 further comprises a plurality of cut-outs: afirst cut-out 128 forming the sample window 109, and a second cut-out129 to provide access to the isolation valve interface 104. As with therecesses which protect the blisters, by providing access to theisolation valve interface 104 only through a cut-out 129 in the housing111, the isolation valve interface 104 is protected to some extent fromaccidental impact, which could actuate the isolation valve and renderthe cartridge inoperable.

1.3.4 Blister Sub-Assembly 112

FIG. 5 shows the blister sub-assembly 112 in more detail. The blistersub-assembly 112 may be manufactured separately, during which theblisters are pre-filled with the liquid reagents necessary forconducting the preferred test, and subsequently adhered to the pneumaticfoil 113.

Blister sub-assemblies (or ‘blister packs’) are familiar to a skilledperson. A blister is a collapsible chamber for containing a liquid,which may be expelled from the blister by pressing on the blister andthereby collapsing it. In typical blister packs, the chamber of ablister is sealed by a foil or other frangible layer which ruptures oncethe pressure inside the chamber reaches a particular magnitude as theblister is collapsed.

In the exemplary cartridge, the blister sub-assembly 112 comprises threeblisters 130 a-c. These contain, respectively, the lysis buffer whichcomprises reagents capable of performing cell lysis, the wash buffer andthe elution buffer.

The exemplary blister sub-assembly 112 comprises a substrate 131 ontowhich the aforementioned blisters 130 a-c are formed by a deformablepolymeric layer which is shaped to provide the chambers. Three apertures132 a-c, corresponding to the three blisters 130 a-c, pass through thesubstrate 132. Each of the apertures is covered by the deformablepolymeric layer forming the chamber, which thereby connects the apertureto the chamber but for a seal 133 a-c between the respective apertures132 a-c and chambers. Upon application of a suitable pressure on theblister 130 a-c, the seal 133 a-c breaks, thereby causing the liquidcontents of the blister to be ejected from the blister and to flowthrough the aperture 132 a-c in the substrate 131 out of the blistersub-assembly.

As shown, the seals 133 a-c at least partially surround the periphery ofthe chambers, where they meet the substrate 131. At the point in eachseal 133 a-c which is designed to break (thereby forming the liquidpassageway between the aperture 132 a-c and chamber), the seal 133 a-cmay be weaker than the rest of the periphery. This ensures that thecorrect part of the seal 133 a-c breaks when the suitable pressure isapplied.

The blisters may be collapsed by the reader when the cartridge isinserted therein. One or more mechanical actuators (such as a foot) maybe applied by the reader into the recess so as to collapse the blister.

The blister sub-assembly 112 further comprises two reference holes 134a-b configured to permit an assembly fixture to provide a reference tofacilitate positioning of the assembly during manufacture.

1.3.5 Pneumatic Layer 114

FIGS. 6A and 6B show the pneumatic layer 114 in more detail. FIG. 6A isa top view of the pneumatic layer and FIG. 6B is a bottom view. Thepneumatic layer 114 is comprised of a rigid plastic layer 135 which, incertain places, is overmoulded with a plurality of flexible membranes toform certain components when the cartridge is assembled. The flexiblemembranes are manufactured from a thermoplastic elastomer.

The rigid plastic layer 135 has a plurality of differently-shapedrecesses therein and apertures therethrough. In combination with thefluidic layer 115, certain recesses within, and/or apertures through,the rigid plastic layer 135 form a number of components, including: thesample mixing chamber 136; the waste chamber 137; the capture column138; the elution chamber 139; the first and second amplificationchambers 140 a-b; and the first to fourth detection chambers 141 a-d. Anaperture 142 is also provided to give access to the electrode layer 117.

In combination with the overmoulded flexible membranes and the pneumaticfoil 113, certain other apertures through the rigid plastic layer form anumber of other components, including: the upstream bellows valve 142;the bellows 143; a pneumatic interface 144; the downstream bellows valve145; the wash buffer inlet valve 146; the wash buffer air inlet valve146 a; the elution buffer inlet valve 147; the elution buffer air inletvalve 147 a; the waste chamber valve 148; the elution chamber valve 149;the isolation valve 150; the first and second amplification chamberinlet valves 151 a-b; and first and second amplification chamber outletvalves 152 a-b. A further aperture, in combination with an overmouldedflexible membrane (but not the pneumatic foil) forms a bypass valve 153.

With the exception of the isolation valve 150 and the bypass valve 153,the valves formed in the pneumatic layer are pneumatically-operablevalves. That is, each valve is operable to open and close a fluidicchannel in which the valve is located, and this valve is actuated byapplying a particular pressure to a pneumatic control line coupled tothe valve. The pneumatic control lines are coupled to the pneumaticinterface 144, to which the reader has access when the cartridge 100 isinserted therein. Hence, to actuate a given pneumatic valve, the readermerely applies an appropriate pressure to the pneumatic control lineassociated with that valve to open or close the valve.

The isolation valve 150 and the bypass valve 153 are also actuated bythe reader, but mechanically. Again, each valve is operable to open andclose a fluidic channel in which the valve is located, but the valve isactuated by applying one or more mechanical actuators (such as a foot)to the valve.

The pneumatic layer further comprises two reference holes 154 a-bconfigured to permit an assembly fixture to provide a reference tofacilitate positioning of the layer during manufacture. When thecartridge is assembled, the reference holes 154 a-b in the pneumaticlayer align with the reference holes 134 a-b in the blistersub-assembly.

The pneumatic layer further comprises apertures 155 a-c which, when thecartridge is assembled, line up with apertures 132 a-c passing throughthe substrate 131 of the blister sub-assembly (through the pneumaticfoil, as described below).

1.3.6 Pneumatic Foil 113

FIG. 7 shows the pneumatic foil 113 in more detail. As explained above,the pneumatic foil 113 is adhered to the upper surface of the pneumaticlayer 114, thereby fluidly sealing channels, chambers, valves, pumps,bellows and other components formed therein. Thus, for the most part,the pneumatic foil 113 is a generally rectangular and planar foil sheetso as to provide an effective seal. Beneficially, the pneumatic foil 113is inert such that is does not react with the reagents which movethrough the pneumatic layer 114.

However, the pneumatic foil 113 does not overlie the entire pneumaticlayer 114. In particular, the pneumatic foil 113 does not overlie thesample mixing chamber 136 or the waste chamber 137 at the non-insertionend 106 of the cartridge 100, or the bypass valve 153 at the insertionend 105. Moreover, the pneumatic foil 113 comprises cut-outs 156, 157,such that it does not overlie the isolation valve 150 or the pneumaticinterface 144, respectively.

The pneumatic foil 113 further comprises three apertures 158 a-c which,when the cartridge 100 is assembled, line up with apertures 132 a-cpassing through the substrate 131 of the blister sub-assembly and 155a-c passing through the pneumatic layer 114. The apertures 158 a-cpermit the liquid reagents within the blisters to pass to the pneumaticlayer 114, and thence to the fluidic layer 115 through apertures 155a-c.

The pneumatic foil 113 comprises two reference holes 159 a-b configuredto permit an assembly fixture to provide a reference to facilitatepositioning of the layer during manufacture. When the cartridge isassembled, the reference holes 159 a-b in the pneumatic foil align withthe reference holes in the other layers.

The pneumatic foil is a composite foil manufactured from a layer ofpolyethylene terephthalate, to provide strength, with a layer ofpolypropylene on top to provide an inert material for contacting theliquid sample and buffers, and also to enable the foil to be heat sealedto the pneumatic layer (also manufactured from polypropylene.

1.3.7 Fluidic Layer 115

FIGS. 8A and 8B show the fluidic layer 115 in more detail. FIG. 8A is atop view of the pneumatic layer and FIG. 8B is a bottom view. Thefluidic layer 115 is comprised of a rigid plastic layer 160. Asexplained previously, the top side of the fluidic layer 115 (not shown)is adhered to the bottom side of the pneumatic layer 113 (see FIG. 5B)such that the various channels, chambers, valves, pumps, bellows andother components formed by a combination of the pneumatic and fluidiclayers are aligned.

As with the rigid plastic layer 135 of the pneumatic layer 113, therigid plastic layer 160 of the fluidic layer 115 has a plurality ofdifferently-shaped recesses therein and apertures therethrough. Incombination with the pneumatic layer 113 and the fluidic foil 116,certain recesses within, and/or apertures through, the rigid plasticlayer 160 forms certain components, including: the sample inlet chamber136; the capture column 138; the elution chamber 139; the first andsecond amplification chambers 140 a-b; and the first to fourth detectionchambers 141 a-d. the upstream bellows valve 142; the bellows 143; thepneumatic interface 144; the downstream bellows valve 145; the washbuffer inlet valve 146; the wash buffer air inlet valve 146 a; theelution buffer inlet valve 147; the elution buffer air inlet valve 147a; the waste chamber valve 148; the elution chamber valve 149; theisolation valve 150; the first and second amplification chamber inletvalves 151 a-b; and first and second amplification chamber outlet valves152 a-b. An aperture 161 is also provided to give access to theelectrode layer 117.

Moreover, in combination with the fluidic foil 116 (but not thepneumatic layer 114), recesses in the fluidic layer 115 also providesthe coarse filter 162, the convoluted mixing channel 163, and aplurality of channels which, when the cartridge is assembled, connectthe aforementioned components together to enable passage of the liquidsample and liquid reagents through the cartridge, and facilitatepneumatic actuation of the valves, pumps, bellows and other components.

The fluidic layer comprises two reference holes 164 a-b configured topermit an assembly fixture to provide a reference to facilitatepositioning of the layer during manufacture. When the cartridge isassembled, the reference holes 164 a-b in the fluidic layer align withthe reference holes in the other layers.

As mentioned above, channels are formed between the pneumatic interfaceand the various valve and bellows described above. In the exemplarycartridge, the pneumatic interface comprises 11 ports which areconnected to the various components as follows.

-   -   Port 1: bellows    -   Port 2: upstream bellows valve    -   first and second amplification chamber inlet valves    -   first and second amplification chamber outlet valves

Port 3: downstream bellows valve

-   -   Port 4: wash buffer inlet valve    -   Port 5: wash buffer air inlet    -   Port 6: wash buffer air inlet valve    -   elution buffer air inlet valve    -   Port 7: elution buffer air inlet    -   Port 8: elution buffer inlet valve    -   Port 9: reference pressure line    -   Port 10: elution chamber valve    -   Port 11: waste chamber valve

It will be understood that whilst various inventive aspects of theexemplary cartridge may be implemented using specific ones of theconnections listed above (in particular, the first and secondamplification chamber inlet and outlet valves being connected to asingle port; and the wash and elution buffer air inlets being connectedto a single port); the precise configuration listed above is notessential.

1.3.8 Fluidic Foil

FIG. 9 shows the fluidic foil 116 in more detail. As explained above,the fluidic foil 116 is adhered to the lower surface of the fluidiclayer 115, thereby fluidly sealing channels, chambers, valves, pumps,bellows and other components formed therein. Thus, for the most part,the fluidic foil 116 is a generally rectangular and planar foil sheet soas to provide an effective seal. Beneficially, the foil 116 is inertsuch that is does not react with the reagents which move in thepneumatic layer.

However, the fluidic foil 116 does not overlie the entire fluidic layer115. In particular, the fluidic foil 116 does not overlie the detectionchambers 141 a-d at the insertion end 105.

The fluidic foil 116 comprises two reference holes 165 a-b configured topermit an assembly fixture to provide a reference to facilitatepositioning of the layer during manufacture. When the cartridge isassembled, the reference holes 165 a-b in the fluidic foil align withthe reference holes in the other layers.

The fluidic foil is a composite foil manufactured from a layer ofpolyethylene terephthalate, to provide strength, with a layer ofpolypropylene on top to provide an inert material for contacting theliquid sample and buffers, and also to enable the foil to be heat sealedto the fluidic layer (also manufactured from polypropylene.

1.3.9 Electrode Layer 117

Finally, FIG. 10 shows the electrode layer 117 in more detail. Asexplained above, the electrode layer 117 is adhered to the fluidic layer115. The electrode layer 117 comprises four sets of detection electrodes166 a-d. Each set of detection electrodes 166 a-d comprises first tothird electrical contacts 168 a-d which couple with correspondingelectrical contacts in the reader when the cartridge is insertedtherein. Preferably, the electrical contacts are made of silver tooptimise the electrical connection. Preferably electrodes which aresilver plated with silver chloride are used to ensure a the optimalgalvanic behaviour.

Each set of detection electrodes 166 a-d comprises a working electrode169 a-d; a counter electrode 170 a-d and a reference electrode 171 a-d.Each of the electrodes is coupled to a respective electrical contact.Each set of detection electrodes 166 a-d also comprises a dielectric 172a-d covering the interface between the electrodes and the respectiveelectrical contacts.

A skilled person understands that electrochemical signalling may be usedto indicate the presence of genetic or immuno targets. In the exemplarycartridge this process is performed in the first to fourth detectionschambers 141 a-d which are optimised to provide the electrochemical testinterface.

The electrodes 166 a-d are arranged such that a liquid sample within thefirst to fourth detection chambers 141 a-d comes into contact with thefirst to fourth sets of electrodes 166 a-d. In the detection chambers,some compounds in the fluid sample (referred to as the ‘electrolyte’)have a natural tendency to migrate to electrodes and swap electrons.This galvanic effect is how batteries work.

All combinations of soluble compounds have some electrochemicalactivity, and the rate at which this activity occurs (i.e. the amount ofcharge exchanged) is determined by exactly what those compounds are.Hence, it is possible to measure the presence of different analytes inthe liquid sample, by searching for characteristic features of theirredox electrochemistry.

In the exemplary cartridge, the current required to maintain a givenredox state in the detection chambers 141 a-d is monitored at differentredox states. Current is supplied through the electrolyte from theworking electrodes 169 a-d to counter electrodes 170 a-d.

The reference electrodes 171 a-d also contact the electrolyte. Voltagesare declared with respect to this reference electrode because itsvoltage is largely independent of the redox conditions and thistherefore means that it is only the redox state of the chemistry at thecontrol electrode that is being measured.

A voltage sweep is applied between the working electrodes 169 a-d andcounter electrodes 170 a-d by the reader, which generates thecharacteristic range of redox conditions. The current passing betweenthe working electrodes 169 a-d and the counter electrodes 170 a-d isthen measured to obtain the test results. The voltage sweep is a slowlyincrementing set of voltages applied between the electrodes. Preferablythe sweep is from about −0.7 volts to about +1 volts relative to thereference electrode. The voltage is applied in consecutive incrementingpulses having a pulse modulation amplitude of between 30 and 80millivolts (preferably between 40 and 60 millivolts; more preferably 50millivolts). Preferably the step increment from one pulse to the next isbetween 1 and 5 millivolts (preferably between 2 and 4 millivolts; morepreferably 3 millivolts). By applying these voltages across theelectrodes, current measurements in the scale of 100s of nano amps maybe obtained.

The particular arrangement of detection electrodes illustrated in FIG.10 may itself form an isolated inventive aspect of the cartridge.Conventionally, the counter electrode in a potentiostat is larger thanthe working electrode to provide an ample supply of surplus electrons.However, it has been found that reversing this convention surprisinglyoffers better results for the exemplary cartridge. For theelectrochemistry performed on the liquid sample described above in theexemplary cartridge, it is found that having a working electrode whichis larger than the counter electrode provides larger signals andimproved results by way of increased sensitivity. In other words, havinga current flow from a relatively large working electrode to a relativelysmall counter electrode offers improvements over the conventionalarrangement.

Preferably each working electrodes 169 a-d is formed in a U-shape andeach counter electrode 170 a-d is formed in a straight elongate shapebetween the two prongs of the respective U-shaped working electrode.

The method operation of the exemplary cartridge introduced above willnow be briefly explained.

1.4 Method of Operation of the Exemplary Cartridge 1.4.1 The Front End

As described above, a fluid sample (such as a urine sample) isintroduced into the sample mixing chamber 10 using a pipette. A portionof the sample passes to the sample indicator 12 to show that a sample ispresent in the sample mixing chamber.

Once the cartridge 100 with a sample in the mixing chamber 10 isinserted into a reader, and the reader is activated, the test maycommence. Firstly, the reader will apply a mechanical actuator (such asa foot) to collapse the lysis buffer blister 14. In doing so, the lysisbuffer will be expelled into the sample mixing chamber 10 where it willmix with the sample.

The bellows 20 and its valves 22 a-b then moves the liquid sample andlysis buffer back and forth into the sample mixing chamber 10 so as tomix the lysis and sample and to rehydrate the internal control.Following the mixing step, incubation of the sample and lysis bufferoccurs to allow cell lysis to take place.

The bellows 20 and its valves 22 a-b will then commence operation topump the sample from the sample mixing chamber 10, into the main channel16, through the coarse filter 18 and toward the capture column 24.Within the capture column 24 nucleic acids are specifically bound to afilter in the capture column on the basis of their size and charge. Theunwanted liquid sample passes through to the waste chamber 38.

Once the unwanted liquid sample has passed to the waste chamber 38,leaving the nucleic acids bound to the capture column 24, the readerapplies a mechanical actuator (such as a foot) to collapse the washbuffer blister 30. In doing so, the wash buffer will be expelled intothe first branch channel 26, and thence into the main channel 16. Again,the bellows 20 and its valves 22 a-b will commence operation to pump thewash buffer through the main channel 16 and through the capture column24 to wash any remaining unwanted cell debris and other cellularcomponents out of the capture column with the wash buffer through to thewaste chamber 38, or else the wash buffer will be flushed into the wastechambers using air from the wash and/or elution buffer air inlets.

Once the wash sample has passed to the waste chamber 38, leaving onlythe bound and purified nucleic acids in the capture column 24, thereader applies a mechanical actuator (such as a foot) to collapse theelution buffer blister 32. In doing so, the elution buffer will beexpelled into the second branch channel 28, and thence into the mainchannel 16. Again, the bellows 20 and its valves 22 a-b will commenceoperation to pump the elution buffer through the main channel 16 andthrough the capture column 24 to elute the nucleic acids from thecapture column, or else the elution buffer will be flushed into thecapture column using air from the wash and/or elution buffer air inlets.The prepared liquid sample then passes through to the elution chamber46; again, either by being pumped or flushed as described above.

The sample settles in the elution chamber 46 allowing bubbles todisperse before entering the amplification chambers.

1.4.2 The Back End

The bellows 20 and its valves 22 a-b will then commence operation topump the liquid sample from the elution chamber 46, through theisolation valve 59, through the mixing channel 52 and into theamplification chambers 56 a-b, or else the sample will be flushed intothe amplification chambers using air from the wash and/or elution bufferair inlets. In the nucleic acid amplification chambers 56 a-d thenucleic acid of interest, if present, is amplified such that it ispresent at a detectable level. The control nucleic acid is alsoamplified such that it is present at a detectable level. As mentionedabove, any nucleic acid amplification method may be used. Where PCR isused, primers specifically hybridise to the nucleic acid of interest andare extended by a thermostable polymerase such as Taq polymerase via theaddition of dNTPs to the 3′ end of each of the primers. Any excessliquid sample may be removed from the fluid pathway through the bypasschannels 68.

The bellows 20 and its valves 22 a-b will then commence operation topump the liquid sample from the amplification chambers 56 a-b and intothe detection chambers 62 a-d, or else the sample will be flushed intothe detection chambers using air from the wash and/or elution buffer airinlets. In the detection chambers, the target probe specificallyhybridises to the target amplified nucleic acid of interest and thecontrol probe specifically hybridises to the amplified control nucleicacid. The nuclease hydrolyses the target and control probes followinghybridisation of the probes to the amplified nucleic acid. Thehydrolysis of the target and control probes frees the labels from theprobes causing a detectable change in the signal from the labels tooccur.

Once the liquid sample occupies the detection chambers, the readerapplies a mechanical actuator to the isolation valve 50 to close thevalve and isolate the liquid sample in the back end of the device.

The electrodes provide a potential difference across the at least onedetection chamber. Depending on the state of the label (i.e. whether itis attached to the full length probe or the probe has been hydrolysedand it is free or attached to a single nucleotide or short part of theprobe), the current that is able to flow through the detection chamberwill differ. The electrodes therefore allow detection by the reader ofthe change in the signal from the label which results from hydrolysis ofhybridised probe.

The present invention will now be described with reference to FIGS. 16to 17.

2. Preventing Reuse of the Cartridge

FIG. 16 shows a top view the exemplary fluidic cartridge 100 describedabove in assembled form. As shown, the cartridge comprises a housing111, in which three openings 112 a-c are provided to gain access tocorresponding recesses. With each recess, a blister 130 a-c of theblister sub-assembly 112 is provided. Thus opening 122 a gives access toblister 130 a (preferably a lysis blister); opening 122 b gives accessto blister 130 b (preferably a wash blister); and opening 122 c givesaccess to blister 130 c (preferably an elution blister).

As described above, each of these blisters may be mechanically actuatedby extending a mechanical actuator (such as a foot) into the recess tocollapse the blister and expel the contents stored therein into afluidic channel.

In the case of the lysis blister, the step of collapsing the blister toexpel the lysis buffer into the sample mixing chamber 10 is a key stepin the diagnostic test being performed on the cartridge. In other words,the diagnostic test cannot start without collapsing the lysis blister130 a and expelling the lysis buffer into the sample mixing chamber 10.Conversely, if the lysis blister 130 a has been collapsed to expel thelysis buffer into the sample mixing chamber 10, then the diagnostic testhas been started and the cartridge cannot be restarted at a later stage.

The cartridge 110 of FIG. 16 comprises various other features andinterfaces described above, but none of these are essential to carryingout the present invention and are therefore not explained here.

FIG. 17 shows a top view of the cartridge of FIG. 16 further comprises alabel 199 overlying the top surface of the housing 111. The label 199 ispreferably adhesively backed for simplicity of manufacture. The label199 is formed of a breakable material such as a thin layer of paper,card, foil or plastic. The label 199 bears various pieces ofinformation, such as the supplier of the cartridge, and various indicia.Ideally, the label is made from a material onto which this informationmay be easily printed.

As can be seen, the label 199 covers the openings 122 a-b in the uppersurface 120 of the housing and thus covers the recesses formed thereintogether with the blisters. In particular, the opening 122 a and recessassociated with the lysis blister 120 a is covered by the label,although all openings and recesses are preferably covered.

The label is breakable. That is, a mechanical actuator, such as a footof the cartridge reader (not shown) is capable of breaking the labelwhen the label is in situ over the openings and recesses. Preferably theforce required to break the label is less than 50 N, preferably lessthan 40 N, preferably less than 30 N, preferably less than 20 N,preferably less than 10 N. The label may be frangible and break bytearing or shattering, or else may deform to some extent prior tobreaking.

Preferably, the portion of the label covering the openings 122 a isperforated to reduce the force required to break that portion of thelabel. Perforations could run across the diameter of the portion fromone side to the other, or extend around the periphery.

Over the portion of the label which covers the opening 122 a and recesscontaining the lysis blister, there is printed a barcode 200. Thebar-code is a machine-readable identification code and may be replacedby, for example, a QR-code or machine readable text. The bar-codecontains information including test type (for example test number ortest name); cartridge lot number and/or cartridge serial number andcartridge expiration date.

As can be seen from FIG. 17, the bar-code occupies a given width w, anda give length I. Since a bar-code reader could read a bar-code with afairly narrow strip of bar-code, the width w of the bar-code is nolarger than the diameter of the opening 122 a such that the bar-code isrendered completely unreadable when a foot having (almost) the samediameter as the opening is inserted into the recess. In otherembodiments, the width w is narrower than the diameter of the opening122 a.

When a cartridge such as that shown in FIG. 17 is inserted into a reader(not shown), preferably the reader attempts automatically to identifythe bar-code. If the cartridge is used (that is, if the lysis blisterhas already been collapsed), the bar-code will have been renderedunreadable and thus the reader will not be able to identify thebar-code. In that situation, the reader will not extend the mechanicalactuator and the test will not start. However, if a bar-code isidentified, the reader may commence a test by extending a mechanicalactuator into the recess to collapse the lysis blister and in theprocess breaking the label 199 thus rendering the bar-code 200unreadable.

The reader may be configured so as to commence the test only if thebar-code (or other machine-readable code) is in a predetermined format.

Of course, the invention may be practiced using machine-readable codesother than bar-codes. The particular disposition of a given code on thelabel to ensure that the code is rendered unreadable by (at least) amechanical actuator having (almost) the same diameter as the openingbeing inserted into the recess will occur to a skilled person based onthe present disclosure.

3. Additional Isolated Inventive Aspects

The following is a non-exhaustive list of isolated aspects of theexemplary cartridge described above which may be claimed. These aspectsare described with reference to FIGS. 11 to 15.

The inclusion of this section does not preclude there being furtheraspects of the exemplary cartridge described above which may also beclaimed.

3.1 Valves for Minimising Dead Volume

An advantageous arrangement for a valve in a fluidic cartridge will nowbe described, which may form an isolated inventive aspect.

Hence, in one aspect, there is provided a valve for a fluidic cartridge,the valve comprising:

-   -   a valve cavity having first and second openings connected to        first and second passageways, respectively; and    -   a flexible membrane movable between a closed position, in which        the flexible membrane seals against the first and second        openings to prevent fluid flow between the first and second        passageways, and an open position, in which the flexible        membrane is spaced apart from the first and second openings to        permit fluid to flow between the first and second passageways;    -   wherein the a valve cavity comprises a roof and a floor, the        floor comprising said first and second openings; and further        comprising:    -   an abutment between the flexible membrane and the roof of the        valve cavity, such that the abutment restricts movement of the        membrane in its open position.

Preferably the abutment is provided on the flexible membrane, andcomprises one or more of a protrusion, a cage, a lip or a crossstructure.

It is sometimes advantageous to limit the extent to which the flexiblemembrane in a valve described herein is able to travel in its openposition. That is, it is desirable to minimise the distance which thevalve membrane moves to its open, and thus minimise the distance it musttravel to close. By minimising this distance, the dead volume within thevalve cavity is reduced, improving the reactivity of the valve.

Hence, as shown in more detail in FIG. 11, preferred embodiments of avalve 300 further comprise an abutment 302. The abutment of theillustrated example is a cross structure, but in different embodimentsmay be a protrusion, cage, lip or similar, attached to the upper surfaceof the flexible membrane 304 so as to contact the roof 306 of the valvecavity and thus limit movement of the membrane in its open position.

It should be appreciated that the channels and openings of the valve arenot shown in FIG. 11.

The abutment is particularly advantageous when filing the amplificationchambers of the exemplary cartridge, because it reduces the dead-volumein the valve cavity and thus limits the distance between the bottomsurface of the flexible membrane and the openings in the valve cavity,thereby permitting a more precise volume of fluid to be metered into theamplification chambers.

3.2 Crack Pressure in Valves

An advantageous arrangement for a valve in a fluidic cartridge will nowbe described, which may form an isolated inventive aspect.

Hence, in one aspect, there is provided a valve for a fluidic cartridge,the valve comprising:

-   -   a valve cavity having first and second openings connected to        first and second passageways, respectively;    -   a flexible membrane within the valve cavity movable between a        closed position, in which the flexible membrane seals against        the first and second openings to prevent fluid flow between the        first and second passageways, and an open position, in which the        flexible membrane is spaced apart from the first and second        openings to permit fluid to flow between the first and second        passageways; wherein    -   the valve is configured such that a pressure required in the        first passageway to move the flexible membrane from the closed        position to the open position is higher than a pressure required        in the second passageway to move the flexible membrane from the        closed position to the open position.

It will be appreciated that within the valve cavity there is a portion(known as the valve chamber) between the flexible membrane and thefloor. There is also a portion within the valve cavity on the other sideof the flexible membrane to the valve chamber. This portion will have avolume. The pressure within that volume may be changed by applying apositive or gauge pressure to the volume via an actuation channel, forexample. The actuation channel may be connected to a source of positiveor gauge pressure via a pneumatic interface, for example. The pressurewithin the volume is known as the actuation pressure. This operation isdescribed in more detail above.

In a preferred arrangement, the first and second openings may bearranged such that fluid in the first passageway acts on the flexiblemembrane only over a relatively small cross-sectional area whereas fluidin the second passageway acts on the flexible membrane over a largercross-sectional area, preferably substantially the whole membrane.

The effect of this is that the valve is able to withstand a much greaterpressure in the first passageway that in the second passageway.

Preferably the valve cavity has a floor comprising the first and secondopenings and one or more walls between which the flexible membraneextends; and wherein the second opening is coupled to a recess in thefloor between the opening and the flexible membrane, the recess having alarger cross-sectional area than the opening.

Preferably the first opening is located centrally within the floor andthe recess extends around the first opening, such that the secondopening is located between the first opening and a wall of the valvecavity. In a particularly preferred arrangement, the valve cavity has acircular cross section, and the recess is an annular recess whichsurrounds the first opening.

Preferably the opening of the second fluid passageway is locatedadjacent the perimeter of the valve chamber. Preferably the valvechamber has a diameter of between 2 and 10 mm, preferably between 3 and7 mm and more preferably 4 and 6 mm. More preferably, the second openingis offset by 2 mm from the first opening.

An exemplary valve is shown in FIG. 12 in its closed position. The valve310 may be used in place of any of the valves of the exemplary fluidiccartridge shown above. The valve comprises a valve cavity 312 having aflexible membrane 314 overlying a cavity floor 316 in which first 318and second 320 apertures are provided, leading to first 322 and second324 fluid passageways, respectively.

The cavity 312 is formed from a void in a first polymer layer(preferably the fluidic layer 114 of the exemplary cartridge) togetherwith a second polymer layer (preferably the second fluidic layer 115 ofthe exemplary cartridge).

The flexible membrane 314 is shown lying across the floor 316 of thecavity such that the valve is shown in its closed position. The valve ismovable from this position to an open position (where it is spaced fromthe floor 316 and the apertures 322, 324 to form a valve chamber), asdescribed herein.

The first opening 318 of the valve is centrally located within theperimeter of the void formed in the first polymer layer, and istherefore centrally located in the valve cavity 312. The second opening324 of the valve is offset from the first opening 322. The secondopening is coupled to an annular recess 326 in the floor, and thus thecross-sectional area over which the fluid in the second passageway 324acts on the flexible membrane 314 is much larger than thecross-sectional area over which the fluid in the first passageway 322acts on the flexible membrane.

The pressure of a fluid in the first passageway acts on the flexiblemembrane only over a relatively small cross-sectional area of theflexible membrane. Thus, because the pressure of a fluid in the valvecavity on the other side of the flexible membrane acts over the wholemembrane, it may be lower without allowing the membrane to move to itsopen position.

In contrast, the pressure of a fluid in the second passageway acts onthe flexible membrane over a relatively large cross-sectional area ofthe flexible membrane. Since the respective cross-sectional areas arecloser, so too is the pressure in the second passageway which theflexible membrane is able to withstand vis-à-vis the pressure in thevalve cavity.

Preferably, the respective cross-sectional areas of the openings of thefluid passageways allows the membrane to resist pressures around 2.5times the actuation pressure on the first, central, fluid passageway,but only pressures equal to the actuation pressure (i.e. the pressure inthe valve cavity) on the opening of the second, offset, fluidpassageway.

3.3 Entry Port Design

An advantageous arrangement for an entry port on a fluidic cartridgewill now be described, which may form an isolated inventive aspect.

Hence, in one aspect, there is provided a fluidic cartridge forprocessing a liquid sample, the cartridge having a sample mixing chambercomprising:

-   -   a sample inlet aperture for introducing a liquid sample into the        sample mixing chamber;    -   a cage surrounding the inlet aperture and extending into the        sample mixing chamber, the cage further comprising one or more        protrusions extending radially inwardly to abut against a sample        delivery device introduced through the sample inlet.

The body of the cage may be formed from one or more elongate bars, orone or more solid walls, depending from the roof of the sample mixingchamber. If solid walls are provided, there is preferably an aperture inthe lower portion of the walls through which a liquid sample introducedby the sample delivery device can pass. Preferably the bars or wallforming the body are tapered to conform to the nib of a conventionalsample delivery device introduced through the sample inlet.

Solid walls have the additional advantage that they provide a barrier toprevent fluid introduced into the mixing chamber from escaping out ofthe inlet aperture, which is particularly useful if the cartridge isturned upside-down during use.

If the cage is formed from solid walls, the protrusion may be a ledgeextending inwardly from the walls leaving an aperture. Preferably theprotrusion extending from the sides of the inlet aperture is positionedabove the floor of the sample mixing chamber; more preferably above aliquid fill level of the sample mixing chamber. This prevents liquidsample from being sucked back into the sample delivery device onceintroduced into the mixing chamber.

Preferably a vent is provided in the sample mixing chamber to allow airto escape from the chamber during the introduction of the sample. Thisis particularly useful when the inlet aperture is sealed by the sampledelivery device.

Preferably a guide channel is provided within the sample mixing chamber(a portion of which is preferably directly underneath the inletaperture) to direct the sample introduced by a sample delivery deviceinto a visual indicator region. An exemplary visual indicator region isdescribed above in connection with the exemplary cartridge.

Preferably a change in refractive index of the visual indicator regiondescribed herein identifies when a sample has been introduced. Thevisual indicator region may comprise a narrow fluid passageway, whichbecomes filled by the fluid sample via capillary action. The filling ofthe narrow fluid passageway changes the refractive index of the visualindicator region and a colour change identifies when a sample has beenintroduced.

A preferred embodiment of this aspect will now be described withreference to the exemplary fluidic cartridge. The housing 111 (see FIG.4) comprises a sample inlet aperture 126 through which a sample may beintroduced into the sample mixing chamber 10 of the cartridge 100 usinga pipette, for example. As shown in more detail in FIG. 13a , the samplemixing chamber 10 is formed from the pneumatic layer 114, which has aroof adjacent the housing 111 in the region of the inlet aperture, and acorresponding inlet aperture through which a sample may be introducedinto the sample mixing chamber 10.

The roof of the mixing chamber 10 comprises a cage structure formed bywalls 330 surrounding the inlet aperture 126 which extend into thesample mixing chamber 10 from the roof, and a ledge 332 extendingradially inwardly from the walls 330. The shape of the cage structureallows a sample delivery device, such as a pipette, to be located in thecorrect position in the sample mixing chamber 10, and the ledge 332prevents the pipette contacting the surfaces of the sample mixingchamber 10, thereby reducing the risk of contamination. The walls 330can be tapered to further increase the engagement with the pipette.

Once the sample delivery device is located through the aperture, theuser can dispense the sample. The ledge 332 is positioned above anominal liquid fill level (not shown) of the sample mixing chamber so asto prevent the user from accidentally sucking the sample back up afterdispensing it into the chamber.

A vent 334 into the chamber is provided to allow air to escape in theevent that the inlet aperture is sealed by the sample delivery device.

A guide 336 is provided within the sample mixing chamber 10, a portionof which is directly underneath the inlet aperture 126 to direct thesample introduced by a sample delivery device into a visual indicatorregion 338. An exemplary visual indicator region is described above inconnection with the exemplary cartridge.

3.4 Thermal Isolation Pockets

An advantageous arrangement for thermal isolation pockets for a nucleicacid amplification chamber on a fluidic cartridge will now be described,which may form an isolated inventive aspect.

In nucleic acid amplification and detection, it is preferable to applyheat evenly throughout the liquid sample. Whilst it is possible to dothis without difficultly in a laboratory by placing heat sourcesequidistantly around the sample, it is much harder to achieve in acartridge.

Hence, in one aspect, there is provided a fluidic cartridge forperforming nucleic acid amplification on a liquid sample, the cartridgecomprising at least one sample processing chamber and a thermallyinsulating region adjacent the chamber to prevent heat loss from thechamber through the thermally insulating region. Preferably the at leastone sample processing chamber is one or both of a nucleic acidamplification chamber and a nucleic acid detection chamber (hence forth‘processing chamber’).

Preferably the nucleic acid processing chamber is adjacent a surface(preferably a bottom surface) of the cartridge for accepting heat froman external source, the chamber situated between the thermallyinsulating region and the surface such that heat passing from theexternal source through the surface and thence the chamber is not lostout of the other side of the chamber owing to the presence of thethermally insulating region. This arrangement is found to make thechange in temperature inside the chamber (for instance when turning theheat source on and off) as fast as possible, which is beneficial forperforming rapid PCR, for example.

This is particularly advantageous because a single heat source may beplaced adjacent the cartridge to supply heat for the amplificationprocess from one side (the heated side), and yet the sample within thecartridge will be heated substantially and the amount of heat lostthrough the unheated side minimised as far as possible.

Preferably the cartridge is comprised of at least a fluidic layer and apneumatic layer in contacting arrangement. The nucleic acid processingchamber may be formed in the fluidic layer and the thermally insulatingregion may be formed in the pneumatic layer. Preferably the fluidiccartridge further comprises a fluidic foil underneath the fluidic layer,the foil forming the aforementioned surface for accepting heat. The useof a thin foil maximises the heat transfer from the external source. Thematerial of the foil may be chosen to optimise the heat transfer. Forinstance, a metal foil may be used, but it is preferred that apolyethylene terephthalate/polypropylene composite is used due to theadvantages in ease of manufacture of the cartridge, together withmaterial strength and acceptable heat transfer properties.

Preferably the thermally insulating region is formed from one or moresealed thermal isolation pockets formed in the pneumatic layer andsealed by a pneumatic foil. The pockets may be filled with gas such asair or may be evacuated during the manufacturing process such that theyprovide a vacuum.

A preferred embodiment of this aspect will now be described withreference to the exemplary fluidic cartridge. As shown in FIG. 3, theexemplary cartridge 100 comprises, from top to bottom, a housing 111, ablister sub-assembly 112, a pneumatic foil 113, a pneumatic layer 114, afluid layer 115 and a fluidic foil 116.

Referring to FIGS. 6A and 6B, which shows the pneumatic layer, sixthermally insulating regions 140 a-b, 141 a-d are provided. Theinsulating regions 140 a-b are located adjacent two correspondingamplification chambers formed in the fluidic layer 115, whilstinsulating regions 141 a-d are located adjacent four correspondingdetection chambers formed in the fluidic layer 115, when the cartridgeis assembled. As shown, the insulating regions 140 a-b consist of aplurality of thermal isolation pockets, whereas insulating regions 141a-d each consist of a single pocket.

During nucleic acid amplification and detection, thermocycling of theamplification and detection chambers takes place. The chambers in thefluidic layer may be heated by applying heat to the bottom of thecartridge 100, adjacent the fluidic layer 115. The thermal isolationpockets retain the heat within the cartridge, minimising heat loss fromthe fluidic layer 115 into the pneumatic layer 114. The thermalisolation pockets also eliminate the need for heating of the fluidicscartridge from both the top and bottom surfaces e.g. heating both thefluidics layer and the pneumatic layer, simplifying the overall designof the cartridge and reader.

The thermal isolation pocket may comprise one large pocket or multiplesmaller pockets. The advantage of using multiple smaller pockets is thatthe risk of convection currents being set up is reduced, thus providingmaximal thermal insulation.

3.5 Capture Column

An advantageous arrangement for a filtering device in a fluidiccartridge (preferably a ‘capture column’) will now be described, whichmay form an isolated inventive aspect.

Hence, in one aspect, there is provided a fluidic cartridge comprising achannel through which a liquid sample may pass, the channel having afilter for capturing biological components and further comprising:

-   -   an upstream portion and a downstream portion; and    -   a capture portion between the upstream and downstream portions        in which the filter is arranged; wherein:    -   the diameter of the capture portion is greater than the diameter        of the upstream and downstream portions.

Preferably the capture portion is a chamber within the channel, thechamber having an inlet surface having an opening coupled to theupstream portion of the channel and an outlet surface having an openingcoupled to the downstream portion of the channel.

Preferably the fluidic cartridge comprises at least two polymer layers,wherein the upstream portion and an upstream part of the capture portionof the channel are formed in a first polymer layer and the downstreamportion and a downstream part of the capture portion of the channel areformed in a second polymer layer; and wherein the filter is clampedbetween the first and second polymer layers.

Preferably the inlet surface of the chamber comprises distributionconduits leading radially outwardly from the opening so as to direct aliquid sample passing through the opening in the inlet surface radiallyoutwardly.

Preferably the outlet surface of the chamber comprises distributionconduits leading radially inwardly toward the opening so as to direct aliquid sample which has passed through the filter radially inwardlytoward the opening in the outlet surface.

A preferred embodiment of this aspect will now be described withreference to the exemplary fluidic cartridge. In the exemplary cartridgedescribed herein, a capture column 24 is provided along the main channel(see FIG. 1). As shown in FIGS. 14a and 14b , the capture column 24 hasfilter 340 which binds DNA from lysed material before releasing itduring elution. As shown in FIG. 14a , capture column 24 comprises aninlet channel 342 leading into a capture chamber 344 at an upstream end346, and an outlet channel 350 leading from capture chamber 344 at adownstream end 348.

A filter 340 is provided in chamber 344, perpendicular to the directionof flow of fluid through the main channel, such that fluid must passthrough filter 340 when passing from the upstream end of the mainchannel 342 to the downstream end 350 of the main channel.

Referring now to FIG. 14b , the inlet and outlet walls (only one isshown) of the chamber comprise distribution conduits 352 configured todirect fluid radially outwardly into the chamber 344 as it enters thechamber, and radially inwardly toward the exit aperture after it haspassed through the filter 340.

3.6 Waste Chamber

An advantageous arrangement for waste chamber in a fluidic cartridgewill now be described, which may form an isolated inventive aspect.

Hence, in one aspect, there is provided a fluidic cartridge comprising achannel through which a liquid sample may pass and a waste chamber forreceiving fluid from the channel, the waste chamber comprising:

-   -   a pipe, coupled to the channel, extending from a bottom surface        of the waste chamber and having an opening elevated above the        bottom surface to pass fluid from the channel into the chamber;        and    -   a vent within the waste chamber configured to vent the waste        chamber to atmosphere.

Preferably the vent comprises a second pipe, coupled to a vent channelwithin the cartridge, extending from the bottom surface of the wastechamber and having an opening elevated above the bottom surface.Preferably the vent passageway comprises at least one Anderson impactor.

Preferably at least one absorbent pad is provided within the wastechamber.

A preferred embodiment of this aspect will now be described withreference to the exemplary fluidic cartridge. In the exemplary cartridgedescribed herein, a waste chamber is provided for collecting and storingwaste fluid which is produced during washing etc. Waste chamber 10 isshown in more detail in FIGS. 15a and 15b . Waste chamber 38 comprises apipe 360, extending substantially vertically from a bottom surface 362of waste chamber 38. The pipe 38 defines a channel having a first end364 connected to the bottom surface of the waste chamber 38 and fluidlyconnected to the main channel 16. A second end 366 of fluid pipe 360 isdisposed within waste chamber 38, and has an opening through which fluidcan flow into the waste chamber.

Preferably, pipe 360 is substantially vertical, and perpendicular to thebottom surface of the waste chamber 38. The opening at the second end ofpipe 360 is located near the top of the waste chamber 38 as shown inFIG. 15b . By providing the first opening near the top of the wastechamber, the risk of leakage is minimised should the cartridge be turnedupside down.

Absorbent pads 368 are also provided in the waste chamber. Preferably,the upper surface of absorbent pads 368 should also be near the top ofwaste chamber 38, even more preferably, the top of absorbent pads 368should be substantially level with the opening at the second end 366.

In the exemplary cartridge described herein, a second opening 370 isprovided in waste chamber 38 as shown in FIG. 15b . The second opening370 is configured to vent main channel 16 via waste chamber 28 toatmospheric pressure. This avoids putting a back pressure along the mainchannel as the waste channel fills with fluid. Preferably, the secondopening 370 is provided at the end of a second pipe 372 protruding fromthe bottom surface of waste chamber 38. The second opening 370 may befluidly connected to a vent passageway (not shown) which has an openingoutside of the cartridge housing to allow the waste chamber to remain atatmospheric pressure. However, venting the waste chamber outside thecartridge carries a small risk of aerosol contamination. To reduce this,the vent path has impact traps and vents under the cartridge cover.

The skilled person will be capable of modifying the exemplary cartridgeto implement the inventive aspects described herein in various waysdepending on circumstances. It is intended that the scope of the presentinvention is defined by the following claims.

1. A disposable fluidic cartridge having a recess therein and amechanically-actuated mechanism disposed within the recess for effectingan operation of the fluidic cartridge; and further comprising: amachine-readable identification code on a breakable label covering atleast a portion of the recess such that the portion of the breakablelabel covering the recess may be broken upon insertion of a mechanicalactuator into the recess to actuate the mechanically-actuated mechanism,thereby rendering the identification code unreadable and preventingreuse of the cartridge.
 2. The disposable cartridge of claim 1, whereinthe actuatable mechanism is a collapsible blister configured to expelits contents into a fluid pathway within the fluidic cartridge as it iscollapsed.
 3. The disposable cartridge of claim 2 for use in diagnostictesting, wherein the collapsible blister contains a liquid component forconducting a diagnostic test.
 4. The disposable cartridge of claim 3,wherein the liquid component is one of a reagent, a lysis buffer, a washbuffer and an elution buffer.
 5. The disposable cartridge of anypreceding claim, wherein the machine-readable identification code is abar-code or a QR-code. 25
 6. The disposable cartridge of any precedingclaim, wherein the machine-readable identification code takes up an areaon breakable label having a length and a width, and wherein at least onethe length and the width of said area is fully contained within theportion of the breakable label covering the recess.
 7. The disposablecartridge of any preceding claim, wherein the portion of the breakablelabel which covers the recess is separated from the remainder of thebreakable label by a perforation to facilitate breaking of the label. 8.A cartridge reader for receiving a disposable fluidic cartridge having arecess, a mechanically-actuated mechanism disposed within the recess foreffecting an operation of the fluidic cartridge, and a machine-readableidentification code on a breakable label covering at least a portion ofthe recess, the reader comprising: a sensor for detecting theidentification code of a disposable cartridge inserted into thecartridge reader; and a mechanical actuator configured extend into therecess to actuate the mechanically-actuated mechanism and break theportion of the breakable label covering the recess thereby rendering theidentification code unreadable and preventing reuse of the cartridge;wherein: the reader is configured only to extend the mechanical actuatorinto a recess of a cartridge inserted into the reader if the sensordetects an identification code on that cartridge.
 9. The cartridgereader of claim 8, wherein the mechanical actuator is a foot configuredto break the portion of the breakable label covering the recess andcollapse a collapsible chamber located within the recess of thecartridge.
 10. The cartridge reader of claim 9, wherein the cartridgereader is configured to perform a diagnostic test on a sample in adisposable cartridge inserted into the reader.
 11. The cartridge readerof any one of claims 8 to 10, wherein the cartridge reader is configuredto activate the sensor to attempt to detect an identification code uponinsertion of a cartridge into the cartridge reader.
 12. A method ofusing a disposable fluidic cartridge having a recess, amechanically-actuated mechanism disposed within the recess for effectingan operation of the fluidic cartridge, and a machine-readableidentification code on a breakable label covering at least a portion ofthe recess, the method comprising: inserting the disposable cartridgeinto a cartridge reader; attempting to detect an identification code onthe breakable label on the disposable cartridge; and, if successful,extending a mechanical actuator into the recess on the disposablecartridge to break the portion of the breakable label covering therecess, thereby rendering the identification code unreadable, andactuate the mechanically-actuated mechanism.
 13. The method of claim 12,wherein the actuatable mechanism is a collapsible blister and whereinthe step of actuating the actuatable mechanism comprises collapsing thecollapsible blister to expel its contents into a fluid pathway withinthe fluidic cartridge.
 14. The method of claim 13, wherein thecollapsible blister contains a liquid component for conducting adiagnostic test, the method further comprising the step of performing adiagnostic test on a sample in the disposable cartridge.
 15. The methodof any one of claims 12 to 14, wherein if the step of attempting todetect an identification code on the breakable label is unsuccessful,the cartridge reader rejects the cartridge.
 16. The method of claim 14,wherein the liquid component is one of a reagent, a lysis buffer, a washbuffer and an elution buffer.
 17. The method of any one of claims 12 to16, wherein the machine-readable identification code is a bar-code or aQR-code.
 18. The method of any one of claims 12 to 17, wherein the stepof attempting to detect an identification code is triggered uponinsertion of the disposable cartridge into the cartridge reader.
 19. Thedisposable cartridge of any preceding claim, comprising polypropylene.20. The disposable cartridge of claim 2 or any claim dependent on claim2, wherein the fluid pathway is formed in a fluidic layer of the fluidiccartridge, said fluidic layer comprising polypropylene.
 21. The fluidiccartridge of any preceding claim, wherein the recess is formed in ahousing of the fluidic cartridge, said housing comprising acrylonitrilebutadiene styrene.